PCSK9 Gene Repressor Fusion Proteins for Specific In Vivo Silencing
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Solution Overview
Problem
Current methods for modulating PCSK9 levels in vivo are ineffective due to off-target effects, genome instability, and lack of safe delivery modalities, necessitating improved gene repressor systems for therapeutic applications.
Innovation Solution
Development of repressor fusion proteins comprising DNA-binding proteins like zinc fingers or catalytically-dead CRISPR proteins linked with repressor domains, along with guide nucleic acids, for targeted transcriptional repression of PCSK9 gene sequences, delivered via vectors and lipid nanoparticles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current methods for modulating PCSK9 levels are used, then cholesterol levels can be reduced, but off-target effects and genome instability occur
Solution Approach 1:
The patent uses catalytically-dead CRISPR proteins (dCas9) as intermediaries that bind to target DNA sequences without causing cleavage. This mediator approach allows specific recruitment of repressor domains to PCSK9 gene loci while avoiding off-target genomic damage, thereby achieving specific PCSK9 repression without the harmful genome instability associated with active CRISPR-Cas systems
Solution Approach 2:
The invention applies local quality by creating fusion proteins where repressor domains (such as KRAB, MeCP2, or DNMT3A) are specifically attached to the DNA-binding domain of dCas9. This localized configuration ensures that repression activity is concentrated precisely at the PCSK9 target site, while the rest of the genome remains unaffected, thus improving specificity without widespread off-target effects
2Reliability
If gene editing approaches are used to modulate PCSK9, then cholesterol clearance can be improved, but genome instability and safety concerns arise
Solution Approach 1:
The patent extracts the catalytic activity from the CRISPR-Cas system by using catalytically-dead dCas9 variants that retain DNA-binding capability but have lost the ability to cleave DNA. This extraction of the harmful cleavage function while preserving the beneficial targeting function allows safe modulation of PCSK9 expression without inducing genome instability or safety concerns associated with permanent genetic modifications
Solution Approach 2:
The invention converts the potential harm of CRISPR-Cas DNA cleavage activity into a benefit by deliberately using catalytically-dead variants. The 'harmful' cleavage function is removed, while the remaining DNA-binding function is harnessed to recruit repressor domains that safely modulate PCSK9 expression, thus transforming a potentially dangerous system into a safe therapeutic tool
3Manufacturing precision
If repressor fusion proteins are used for PCSK9 targeting, then specific repression can be achieved, but delivery to target cells remains challenging
Solution Approach 1:
The patent employs lipid nanoparticles (LNPs) as universal delivery vehicles that can transport repressor fusion proteins to various target cell types, including hepatocytes. This multi-functional delivery system addresses the challenge of cellular uptake and intracellular delivery, enabling precise PCSK9 repression in liver cells while maintaining ease of administration through intravenous injection
Data Source
AI summary
Provided herein are gene repressor systems comprising fusion proteins, such as fusion proteins comprising a DNA binding domain such as a TALE, zinc finger or catalytically-dead CRISPR protein and guide nucleic acid (gRNA), which are useful in the repression of a proprotein convertase subtilisin/kexin Type 9 (PCSK9) gene. Also provided are methods of using such systems to repress transcription of PCSK9.